One of the most consistently and severely affected macromolecular complexes in neurons from individuals with Alzheimer's disease (AD) is the neuronal cytoskeleton. Elements of the neuronal cytoskeleton are found in neurofibrillary tangles (NFTs), neuritic senile plaques (SPs) and in neuropil threads (NTs), all hallmarks of AD. To date, however, the role of cytoskeletal proteins in the pathogenesis of neurodegenerative diseases remains incompletely defined. The filamentous pathology (e.g., NFTs) found in brains from AD and normal aging may be an epiphenomenon of neuronal death, or a direct contributor to neuron dysfunction and degeneration. In this regard, further investigation of the cytoskeletal proteins thought to comprise these abnormal structures is mandated to determine how these proteins assemble into a dynamic and integrated network in vitro that, subsequently, may provide clues to their in vivo interactions. Understanding the assembly properties of structural proteins implicated in cytoskeletal pathology may enable us to discern how and why these proteins form NFTs. With this information, the role of the NFT in neurodegeneration may then be clarified. This grant proposal focuses on the assembly properties of the neurofilaments (NF-H, NF-M, NF-L) and the recently described intermediate filament, alpha-internexin/ NF 66kD protein, and how these proteins in combination with microtubules (MTs) and microtubule associated proteins (MAPs) assemble in vitro. Our working hypothesis is that upon isolating, purifying and reassembling these proteins from the CNS of AD subjects and normal age-matched controls, we can determine specific properties that dictate their ability to form normal or abnormal structures. Therefore our experiments may provide new information of cytoskeletal interactions that may precipitate abnormal structural formations leading to neuronal degeneration and cell death.